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Western blot analysis of Peroxisomal Membrane Protein 70 (PMP70) was performed by loading the following whole cell extracts: 20 µg Rat Kidney (Lane 1), 20 µg Rat Liver (Lane 2), 20 µg Mouse Lung (Lane 3), 10 µg A431 cells (Lane 4), and 10 µg U2OS cells (Lane 5) per well onto a 4-20% Bis-Tris polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and the membrane was probed with Pierce Fast Western Kit (Product # 35050). The membrane was probed with PMP70 Polyclonal antibody (Product # PA1-650) at a dilution of 1:500 for 30 minutes at room temperature on a rocking platform per Fast Western Kit instructions. The membrane was then washed in kit wash buffer and probed with kit secondary antibody reagent for 10 minutes (diluted at 150 µL per 10 mL kit reagent diluents). The membrane was then washed in kit wash buffer and chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

Western blot analysis of PMP70 was performed by loading 25 µg of mouse lung (lane 1), mouse liver (lane 2) and HepG2 (lane 3) cell lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4°C overnight. The membrane was probed with a PMP70 polyclonal antibody (Product # PA1-650) at a dilution of 1:1000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at ~70 kDa.

Flow cytometry analysis of PMP70 in NIH-3T3 cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a PMP70 polyclonal antibody (Product # PA1-650) at a dilution of 0.5 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Flow cytometry analysis of PMP70 in HepG2 cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a PMP70 polyclonal antibody (Product # PA1-650) at a dilution of 0.5 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Flow cytometry analysis of PMP70 in 293T cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a PMP70 polyclonal antibody (Product # PA1-650) at a dilution of 0.5 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Product Specific Information

PA1.1-650 has been successfully used in Western blot, ICC/IF and FACS procedures. By Western blot, this antibody detects an ~70 kDa protein representing PMP70 from mouse kidney protein extracts.

The PA1.1-650 immunogen is a synthetic peptide corresponding to residues C N(644) Y E F K K I T E D T V E F G S(659) of rat PMP70. This sequence is completely conserved in mouse PMP70 and differs from human PMP70 by a single amino acid substitution. PA1.1-650 immunizing peptide (Cat. # PEP-038) is available for use in neutralization and control experiments.

Target Information

Peroxisomes are single membrane organelles which are involved with many important biochemical pathways and are found in almost all eukaryotic cells. Various toxins including, phenols, alcohols, and aldehydes are targeted for modification by peroxisomes. Long chain fatty acid metabolism via beta-oxidation is also mediated by this organelle. Loss of peroxisomal functions may result in diseases such as Zellweger syndrome, hyperpipecolic acidemia, neonatal adrenoleukodystrophy, and infantile Refsum's disease. Peroxisomal membrane protein 70 (PMP70) is an abundant, integral membrane protein of the peroxisome. This protein is induced by treatment with hypolipidemic agents in parallel with peroxisome proliferation and stimulation of the peroxisomal beta-oxidation enzymes.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.